Agent-based approach for elucidating the release from collective arrest of cell motion in corneal epithelial cell sheet.

Changes in cell fluidity have been observed in various cellular tissues and are strongly linked to biological phenomena such as self-organization. Recent studies suggested variety of mechanisms and factors, which are still being investigated. This study aimed to investigate changes in cell fluidity in multi-layered cell sheets, by exploring the collective arrest of cell motion and its release in cultures of corneal epithelial cells. We constructed mathematical models to simulate the behaviors of individual cells, including cell differentiation and time-dependent changes in cell-cell connections, which are defined by stochastic or kinetic rules. Changes in cell fluidity and cell sheet structures were expressed by simulating autonomous cell behaviors and interactions in tissues using an agent-based model. A single-cell level spatiotemporal analysis of cell state transition between migratable and non-migratable states revealed that the release from collective arrest of cell motion was initially triggered by a decreased ability to form cell-cell connections in the suprabasal layers, and was propagated by chain migration. Notably, the disruption of cell-cell connections and stratification occurred in the region of migratable state cells. Hence, a modeling approach that considers time-dependent changes in cell properties and behavior, and spatiotemporal analysis at the single-cell level can effectively delineate emergent phenomena arising from the complex interplay of cells.

Safety of Intravenous Autologous Bone Marrow-Derived Mesenchymal Cell Transplantation in 5 Patients With Reduced Left Ventricular Ejection Fraction.

Background: Although intracardiac injection or intracoronary delivery of mesenchymal stem cells (MSCs) has been reported, there have been few studies on the intravenous injection of MSCs, particularly in Japan. Methods and Results: Five patients with left ventricular ejection fraction (LVEF) ≤45% received 1.0×108 MSCs intravenously. The procedure did not induce significant changes in vital signs. One patient had an elevated body temperature after 1 day, but recovered spontaneously. Laboratory tests remained normal for 1 month after cell delivery. Computed tomography was performed after 1-2 years, and there was no evidence of malignancy. Conclusions: In this pilot study of patients with reduced LVEF, intravenous MSC delivery had no adverse effects.

Bone formation potential of collagen type I-based recombinant peptide particles in rat calvaria defects.

INTRODUCTION: This study aimed to examine the bone-forming ability of medium-cross-linked recombinant collagen peptide (mRCP) particles developedbased on human collagen type I, contains an arginyl-glycyl-aspartic acid-rich motif, fabricated as bone filling material, compared to that of the autologous bone graft. METHODS: Calvarial bone defects were created in immunodeficient rats though a surgical procedure. The rats were divided into 2 groups: mRCP graft and tibia bone graft (bone graft). The bone formation potential of mRCP was evaluated by micro-computed tomography and hematoxylin-eosin staining at 1, 2, 3, and 4 weeks after surgery, and the data were analyzed and compared to those of the bone graft. RESULTS: The axial volume-rendered images demonstrated considerable bony bridging with the mRCP graft, but there was no significant difference in the bone volume and bone mineral density between the mRCP graft and bone graft at 4 weeks. The peripheral new bone density was significantly higher than the central new bone density and the bottom side score was significantly higher than the top side score at early stage in the regenerated bone within the bone defects. CONCLUSION: These results indicate that mRCP has a high potential of recruiting osteogenic cells, comparable to that of autologous bone chips.

Cell jamming, stratification and p63 expression in cultivated human corneal epithelial cell sheets.

Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies. Here we report an investigation into the quality of cultivated human corneal epithelial cell sheets using time-lapse imaging of the cell culture process every 20 minutes over 14 days to ascertain the level of cell jamming, a phenomenon in which cells become smaller, more rounded and less actively expansive. In parallel, we also assessed the expression of p63, an important corneal epithelial stem cell marker. The occurrence of cell jamming was variable and transient, but was invariably associated with a thickening and stratification of the cell sheet. p63 was present in all expanding cell sheets in the first 9 days of culture, but it's presence did not always correlate with stratification of the cell sheet. Nor did p63 expression necessarily persist in stratified cell sheets. An assessment of cell jamming, therefore, can shed significant light on the quality and regenerative potential of cultivated human corneal epithelial cell sheets.

Extemporaneous Preparation of Injectable and Enzymatically Degradable 3D Cell Culture Matrices from an Animal-Component-Free Recombinant Protein Based on Human Collagen Type I.

Injectable hydrogels are considered important to realize safe and effective minimally invasive therapy. Although animal-derived natural polymers are well studied, they typically lack injectability and fail to eliminate the potential risks of immunogenic reactions or unknown pathogen contamination. Despite extensive research activities to explore ideal injectable hydrogels, such state-of-the-art technology remains inaccessible to non-specialists. In this article, the design of a new injectable hydrogel platform that can be extemporaneously prepared from commercially available animal-component-free materials is described. The hydrogels can be prepared simply by mixing mutually reactive aqueous solutions without necessitating specialized knowledge or equipment. Their solidification time can be adjusted by choosing proper buffer conditions from immediate to an extended period of time, that is, few or several tens of minutes depending on the concentration of polymeric components, which not only provides injectability, but enables 3D encapsulation of cells. Mesenchymal stromal/stem cells can be encapsulated and cultured in the hydrogels at least for 2 weeks by traditional cell culture techniques, and retrieved by collagenase digestion with cell viability of approximately 80%. This hydrogel platform accelerates future cell-related research activities.

The Therapeutic Effects of Adipose-Derived Stem Cells and Recombinant Peptide Pieces on Mouse Model of DSS Colitis.

Cell therapies using adipose-derived stem cells (ADSCs) have been used to treat inflammatory bowel disease (IBD) in human and dog. We previously reported the CellSaic technique, which uses a recombinant scaffold to enhance the efficacy of cell therapy. To examine whether this technique can be applied to cell therapy for colitis, we evaluated the efficacy of CellSaic in colitis mouse models. Colitis mouse models were developed by administering dextran sulfate sodium (DSS) to C57BL/6 mice for 7 days. Then CellSaic comprising human/canine ADSCs (1.2 × 106 cells) or human/canine ADSCs only (1.2 × 106 cells) were administered to the mice. The body weights were measured, and the colon length measurements and histological evaluations were conducted at 7 days after administration. After in vitro culture of human ADSC (hADSC) CellSaic and hADSC spheroids in medium containing TNFα, the levels of the anti-inflammatory protein TSG-6 in each supernatant were measured. Furthermore, we conducted tumorigenicity and general toxicity tests of canine ADSC (cADSC) CellSaic in NOG mice for 8 weeks. In the colitis mouse models, the ADSC CellSaic group presented recovery of body weight and colon length compared with the ADSC-only group. Histological analysis showed that ADSC CellSaic decreased the number of inflammatory cells and repaired ulceration. In vitro, hADSC CellSaic secreted 3.1-fold more TSG-6 than the hADSCs. In addition, tumorigenicity and general toxicity of cADSC CellSaic were not observed. This study suggests that human and canine ADSC CellSaic has a therapeutic effect of colitis in human and dogs.

Molecular characteristics of the KCNJ5 mutated aldosterone-producing adenomas.

The pathophysiology of aldosterone-producing adenomas (APAs) has been investigated via genetic approaches and the pathogenic significance of a series of somatic mutations, including KCNJ5, has been uncovered. However, how the mutational status of an APA is associated with its molecular characteristics, including its transcriptome and methylome, has not been fully understood. This study was undertaken to explore the molecular characteristics of APAs, specifically focusing on APAs with KCNJ5 mutations as opposed to those without KCNJ5 mutations, by comparing their transcriptome and methylome status. Cortisol-producing adenomas (CPAs) were used as reference. We conducted transcriptome and methylome analyses of 29 APAs with KCNJ5 mutations, 8 APAs without KCNJ5 mutations and 5 CPAs. Genome-wide gene expression and CpG methylation profiles were obtained from RNA and DNA samples extracted from these 42 adrenal tumors. Cluster analysis of the transcriptome and methylome revealed molecular heterogeneity in APAs depending on their mutational status. DNA hypomethylation and gene expression changes in Wnt signaling and inflammatory response pathways were characteristic of APAs with KCNJ5 mutations. Comparisons between transcriptome data from our APAs and that from normal adrenal cortex obtained from the Gene Expression Omnibus suggested similarities between APAs with KCNJ5 mutations and zona glomerulosa. The present study, which is based on transcriptome and methylome analyses, indicates the molecular heterogeneity of APAs depends on their mutational status. Here, we report the unique characteristics of APAs with KCNJ5 mutations.

Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration.

We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000011929 .).

Antifibrotic effect of pirfenidone in a mouse model of human nonalcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis with lobular inflammation and hepatocyte injury. Pirfenidone (PFD) is an orally bioavailable pyridone derivative that has been clinically used for the treatment of idiopathic pulmonary fibrosis. However, it remains unknown whether PFD improves liver fibrosis in a mouse model with human NASH-like phenotypes. In this study, we employed melanocortin 4 receptor-deficient (MC4R-KO) mice as a mouse model with human NASH-like phenotypes to elucidate the effect and action mechanisms of PFD on the development of NASH. PFD markedly attenuated liver fibrosis in western diet (WD)-fed MC4R-KO mice without affecting metabolic profiles or steatosis. PFD prevented liver injury and fibrosis associated with decreased apoptosis of liver cells in WD-fed MC4R-KO mice. Pretreatment of PFD inhibited the tumor necrosis factor-α (TNF-α)-induced liver injury and fibrogenic responses associated with decreased apoptosis of liver cells in wild-type mice. PFD also prevented TNF-α-induced hepatocyte apoptosis in vitro with reduced activation of caspase-8 and -3. This study provides evidence for the antifibrotic effect of PFD in a mouse model of human NASH. The data of this study highlight hepatocyte apoptosis as a potential therapeutic target, and suggest that PFD can be repositioned as an antifibrotic drug for human NASH.

Expression of inflammation-related genes in aldosterone-producing adenomas with KCNJ5 mutation.

BACKGROUND: The adrenocortical cells have been shown to produce various inflammatory cytokines such as TNFα and IL-6, which could modulate steroidogenesis. However, the role of inflammatory cytokines in aldosterone-producing adenomas (APAs) is not fully understood. In the present study, we examined the relationships between mRNA expression levels of the inflammation-related genes and somatic mutations in APA tissues. METHODS: We evaluated mRNA expression levels of TNFA, IL6, and NFKB1 in APA tissues obtained from 44 Japanese APA patients. RESULTS: We revealed that mRNA expression patterns of the inflammation-related genes depended on a KCNJ5 somatic mutation. In addition, we showed that mRNA expression levels of the inflammation-related genes correlated with those of the steroidogenic enzyme CYP11B1 in the patients with APAs. CONCLUSION: The present study documented for the first time the expression of inflammation-related genes in APAs and the correlation of their expression levels with the KCNJ5 mutation status and mRNA expression levels of steroidogenic enzymes, indicating the pathophysiological relevance of inflammation-related genes in APAs.

Integration of transcriptome and methylome analysis of aldosterone-producing adenomas.

OBJECTIVE: The pathophysiology of aldosterone-producing adenomas (APA) has been investigated intensively through genetic and genomic approaches. However, the role of epigenetics in APA is not fully understood. In the present study, we explored the relationship between gene expression and DNA methylation status in APA. METHODS: We conducted an integrated analysis of transcriptome and methylome data of paired APA-adjacent adrenal gland (AAG) samples from the same patient. The adrenal specimens were obtained from seven Japanese patients with APA who underwent adrenalectomy. Gene expression and genome-wide CpG methylation profiles were obtained from RNA and DNA samples that were extracted from those seven paired tissues. RESULTS: Methylome analysis showed global CpG hypomethylation in APA relative to AAG. The integration of gene expression and methylation status showed that 34 genes were up-regulated with CpG hypomethylation in APA. Of these, three genes (CYP11B2, MC2R, and HPX) may be related to aldosterone production, and five genes (PRRX1, RAB38, FAP, GCNT2, and ASB4) are potentially involved in tumorigenesis. CONCLUSION: The present study is the first methylome analysis to compare APA with AAG in the same patients. Our integrated analysis of transcriptome and methylome revealed DNA hypomethylation in APA and identified several up-regulated genes with DNA hypomethylation that may be involved in aldosterone production and tumorigenesis.

Recovery process of sciatic nerve defect with novel bioabsorbable collagen tubes packed with collagen filaments in dogs.

Autologous nerve graft is the most commonly applied treatment for the patients with peripheral nerve defect, while application is limited because of tissue availability and unfavorable donor site morbidity. To overcome this problem, peripheral nerve regeneration using a nerve conduit has been studied. Especially, nerve conduit using biodegradable materials has been considered promising. In this study, a potential of collagen nerve conduit has been studied with special reference to the regenerating process of a peripheral nerve. Twelve adult female Beagle dogs weighting 10-12 kg were used. The peroneal nerve was cut to make a 30-mm defect. The nerve defect was bridged by the collagen artificial nerve conduit. Comprehensive functional, electrophysiological, morphometrical, and histological analyses were performed until one year after operation. The wet weight of tibialis anterior muscles was only 32.4% of the healthy side at 24 weeks, which was recovered to 77.4% at 52 weeks after denervation. Electrophysiological evaluation of tibialis anterior muscle belly showed polyphasic wave at 52 weeks after implantation, which was almost half amplitude as compared with that of control. The diameters of myelinated nerve fibers thickened day by day, and the average diameter was 5.16 microm at PFN, 3.91 microm at CG, and 3.75 microm at DFN, and average thickness of myelin sheath was 0.94 microm at PFN, 0.46 microm at CG, and 0.55 microm at DFN after 52 weeks. The distribution of myelinated nerve fiber size in the 52 weeks group was distinctly bimodal with the major peak at approximately 2-4 microm and the minor peak at 10-12 microm. These findings were consistent with the distribution of the normal nerve fiber. This study proves the feasibility of the collagen artificial nerve conduit for promoting nerve regeneration, raises new possibilities of seeking alternatives to autograft for nerve repair. The results from this study showed detailed process of morphological, electrophysiological, and functional recovery of the regenerated nerve, which would provide scientific background for this novel therapy.

Quiescent epithelial cell rests of Malassez can differentiate into ameloblast-like cells.

Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of Hertwig's epithelial root sheath (HERS) that are involved in the formation of tooth roots. After completion of crown formation, HERS are converted from cervical loop cells, which have the potential to generate enamel for tooth crown formation. Cervical loop cells have the potential to differentiate into ameloblasts. Generally, no new ameloblasts can be generated from HERS, however this study demonstrated that subcultured ERM can differentiate into ameloblast-like cells and generate enamel-like tissues in combination with dental pulp cells at the crown formation stage. Porcine ERM were obtained from periodontal ligament tissue by explant culture and were subcultured with non-serum medium. Thereafter, subcultured ERM were expanded on 3T3-J2 feeder cell layers until the tenth passage. The in vitro mRNA expression pattern of the subcultured ERM after four passages was found to be different from that of enamel organ epithelial cells and oral gingival epithelial cells after the fourth passage using the same expansion technique. When subcultured ERM were combined with subcultured dental pulp cells, ERM expressed cytokeratin14 and amelogenin proteins in vitro. In addition, subcultured ERM combined with primary dental pulp cells seeded onto scaffolds showed enamel-like tissues at 8 weeks post-transplantation. Moreover, positive staining for amelogenin was observed in the enamel-like tissues, indicating the presence of well-developed ameloblasts in the implants. These results suggest that ERM can differentiate into ameloblast-like cells.

Tissue-engineered cultured periosteum used with platelet-rich plasma and hydroxyapatite in treating human osseous defects.

BACKGROUND: The aim of the present controlled clinical study was to compare the clinical response of human cultured periosteum (HCP) sheets in combination with platelet-rich plasma (PRP) and porous hydroxyapatite (HA) granules to a mixture of PRP and HA in the treatment of human infrabony periodontal defects. METHODS: Thirty interproximal infrabony osseous defects in 30 healthy, non-smoking subjects diagnosed with chronic periodontitis were included in this study. The subjects were randomly assigned to the test group (HCP sheets combined with PRP and HA) or the control group (PRP with HA). Clinical and radiographic measurements were made at baseline and the 12-month post-surgical evaluation. RESULTS: Compared to baseline, the 12-month results indicated that both treatment modalities resulted in statistically significant changes (P <0.01) in the gingival index, bleeding on probing, probing depth, clinical attachment level, and radiographic infrabony defect depth. Compared to the control group, the test group exhibited a statistically significantly more favorable change in clinical attachment gain (3.9 +/- 1.6 mm versus 2.7 +/- 1.3 mm; P <0.05), vertical relative attachment gain (83.5% +/- 31.7% versus 55.0% +/- 21.9%; P <0.05), and radiographic infrabony defect fill (4.9 +/- 1.2 mm versus 3.2 +/- 1.1 mm; P <0.01). CONCLUSIONS: Compared to PRP with HA, treatment with a combination of HCP sheets, PRP, and HA led to a significantly more favorable clinical improvement in infrabony periodontal defects. A factor likely contributing to these favorable clinical results is the presence of osteogenic cells in the HCP sheets, which provided greater regeneration potential.

A novel approach to regenerating periodontal tissue by grafting autologous cultured periosteum.

In the field of oral and maxillofacial surgery, tissue-engineering techniques have been found useful in regenerating lost tissues. Periodontal disease causes severe destruction of periodontal tissue, including the alveolar bone. In this study we attempted to regenerate canine periodontal tissue defects by grafting autologous cultured membrane derived from the periosteum. Under appropriate culture conditions, periosteal cells produce enough extracellular matrix to form sheets. Periosteum specimens were peeled from the mandibular body of adult hybrid dogs and were cultured until cells formed membrane. ALP activity was measured to determine an optimal time for grafting. The cultured periosteum (CP) was grafted and sutured on a mechanically made Class III furcation defect in the 4th mandibular premolars. After 3 months, the samples were harvested and observed radiologically and histologically. In cases of CP, the bone defects were regenerated and filled with newly formed hard tissue, whereas in the controls the defects remained. These results show that our novel treatment is effective in regenerating alveolar bone for the treatment of periodontal disease.

Fabrication of cultured oral gingiva by tissue engineering techniques without materials of animal origin.

BACKGROUND: Cultured gingival substitute has been found to be a useful graft material for treatment of gingival recession. However, such substitutes include xenograft derivative materials that involve concomitant risk of viral contamination. To eliminate this risk, we designed new gingival substitutes made of recombinant human collagen types I and III sponges and cultured these substitutes in animal-free media (HFDM-1). METHODS: Gingival fibroblasts were seeded onto sponges of type I or III recombinant collagen. These sponges were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), HFDM-1 with 2% human serum (HS), or HFDM-1. Fibroblast proliferation in these samples was compared using the cell-counting kit assay. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released into the cultured media were examined by enzyme-linked immunosorbent assay. RESULTS: The fibroblasts proliferated significantly in all six combinations of collagen and medium types. The fibroblast growth rate after 9 days of culture was equal between HFDM-1 with 2% HS and DMEM with 10% FBS. The type III collagen sponge showed a higher fibroblast growth rate than the type I sponge. VEGF concentrations in HFDM-1 with 2% HS were higher than those in other media. The highest HGF levels were detected in DMEM with 10% FBS. CONCLUSIONS: The new cultured gingival substitute containing no animal-derived materials produced good cell proliferation and VEGF release. The results suggested that the substitute may provide a new tool for the treatment of gingival recession.

Constructing a tissue-engineered ureter using a decellularized matrix with cultured uroepithelial cells and bone marrow-derived mononuclear cells.

This study investigated the efficacy of the ureteral decellularized matrix (UDM) as a scaffold material for a tissue-engineered ureter, and the effect of bone marrow-derived mononuclear cells (BM-MNC) on the neovascularization of the scaffold. Canine ureters were treated with deoxycholic acid to remove all cells. Uroepithelial cells (UEC) were obtained from canine bladders, cultured, and then seeded onto the inner surface of the UDM before transplantation into the subcutaneous space of nude mice or the omentum of nude rats. The cultured UECs began showing vacuolar degeneration 3 days after transplantation and gradually disappeared thereafter. To facilitate neovascularization in the implant, BM-MNCs were seeded around the UDM before transplantation. This facilitated the survival of the UECs, which formed three to five cellular layers after 14 days. The mean microvessel density was significantly increased in tissues seeded with BM-MNCs. However, cell-tracking experiments revealed that the increased number of capillaries in the experimental group was not due to the direct differentiation of transplanted endothelial progenitor cells. Our results demonstrate that the UDM is a useful scaffold for a tissue-engineered ureter, especially when seeded with BM-MNCs to enhance angiogenesis.

Ultrasound enhances transforming growth factor beta-mediated chondrocyte differentiation of human mesenchymal stem cells.

In clinical studies and animal models, low-intensity ultrasound (US) promotes fracture repair and increases mechanical strength. US also promotes cartilage healing by increasing glycosaminoglycan synthesis of chondrocytes. As mesenchymal stem cells (MSCs) have the ability to differentiate into chondrocytes, US may promote their differentiation. Here, we evaluated the effects of US on the differentiation of MSCs toward chondrocytes and cartilage matrix formation. When human MSCs cultured in pellets were treated with transforming growth factor beta (TGF-beta, 10 ng/mL), they differentiated into chondrocytes as assessed by alcian blue staining and immunostaining for aggrecan, but nontreated cell pellets did not. Furthermore, when low-intensity US was applied for 20 min every day to the TGF-beta-treated cell pellets, chondrocyte differentiation was enhanced. Biochemically, aggrecan deposition was increased by 2.9- and 8.7-fold by treatment with TGF-beta alone, and with both TGF-beta and US, respectively. In contrast, cell proliferation and total protein amount appeared unaffected by these treatments. These results indicate that low-intensity US enhances TGF-beta-mediated chondrocyte differentiation of MSCs in pellet culture and that application of US may facilitate larger preparations of chondrocytes and the formation of mature cartilage tissue.

Autogenous injectable bone for regeneration with mesenchymal stem cells and platelet-rich plasma: tissue-engineered bone regeneration.

We have attempted to regenerate bone in a significant osseous defect with minimal invasiveness and good plasticity, and to provide a clinical alternative to autogenous bone grafts. Platelet-rich plasma (PRP) may enhance the formation of new bone and is nontoxic, nonimmunoreactive, and accelerates existing wound-healing pathways. We have used a combination of PRP as an autologous scaffold with in vitro-expanded mesenchymal stem cells (MSCs) to increase osteogenesis, compared with using the scaffold alone or autogenous particulate cancellous bone and marrow (PCBM). The newly formed bones were evaluated by radiography, histology, and histomorphometric analysis in the defects at 2, 4, and 8 weeks. According to the histological observations, the dog MSCs (dMSCs)/PRP group had well-formed mature bone and neovascularization compared with the control (defect only), PRP, and PCBM groups at 2 and 4 weeks. Histometrically, at 8 weeks newly formed bone areas were 18.3 +/- 4.84% (control), 29.2 +/- 5.47% (PRP), 61.4 +/- 3.38% (PCBM), and 67.3 +/- 2.06% (dMSCs/PRP). There were significant differences between the PCBM, dMSCs/PRP, and control groups. These results demonstrate that the dMSCs/PRP mixture is useful as a osteogenic bone substitute.

Differential regulation by IL-1beta and EGF of expression of three different hyaluronan synthases in oral mucosal epithelial cells and fibroblasts and dermal fibroblasts: quantitative analysis using real-time RT-PCR.

Using "real-time RT-PCR", we assessed the expression of three different hyaluronan synthase genes, HAS1, HAS2, and HAS3, by measuring their mRNA amounts in cultured human oral mucosal epithelial (COME) cells, oral mucosal fibroblasts, and dermal fibroblasts, and investigated the effects of interleukin-1beta (IL-1beta) and epidermal growth factor (EGF). When COME cells were treated with IL-1beta or EGF, early and marked increases and subsequent rapid decreases were observed for all HAS genes and, moreover, actual changes in hyaluronan synthesis subsequently occurred. The effects of IL-1beta stimulation were concentration-dependent and the maximal response to the EGF stimulation was observed at a low concentration (0.1 ng per mL). When two different types of fibroblasts were treated with IL-1beta or EGF, increased expression with different degrees and rates of three different HAS genes and subsequent increased synthesis of hyaluronan were also observed. In addition, HAS1 gene expression was not detectable in the mucosal fibroblasts, while weak HAS3 gene expression was detected in the dermal fibroblasts. Taken together, it is likely that the regulation of the expression of the three different HAS genes is different between oral mucosa and skin, which may be of significance for elucidating some of the differences between these tissues in wound healing.